MINISTRY OF AGRICULTURE, FISHERIES AND FOOD CSG 15 Research and Development Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit MAFF, Area 6/01 1A Page Street, London SW1P 4PQ An electronic version should be e-mailed to [email protected]

Project title Characterisation and pathogenesis of epizootics in wild freshwater (Phase II)

MAFF project code FC1106 Contractor organisation and location CEFAS Weymouth Laboratory, Barrack Road, Weymouth, Dorset. DT4 8UB

Total MAFF project costs £

Project start date 01/04/96 Project end date 31/03/00

Executive summary (maximum 2 sides A4)

This contract provided information on a number of fish disease issues including the assessment of epizootics affecting wild fish populations and the possibility of introduction of exotic parasites via the importation of cold water ornamental fish.

Epizootics amongst cyprinid fry caused by myxosporean parasites were investigated in liaison with the Environment Agency (EA). Several thousand fry, mainly roach (Rutilus rutilus), chub (Leuciscus cephalus), dace (Leuciscus leuciscus) and minnow ( phoxinus) were collected and examined histologically for myxosporean infections. The main parasite of concern has been identified as Myxobolus cyprini, which primarily affects the muscle tissues. Chub appear to be most susceptible and based on the pathological response, fish with heavy infections are certainly debilitated and some are unlikely to survive. However, levels of infection fluctuate from year to year and between different rivers sampled in the same year. Chub fry from certain locations also exhibit skeletal deformities and these were found to be associated with another myxosporean parasite. Whether affected fish survive is at present uncertain although occasional adult fish exhibiting skeletal deformity do occur.

In liaison with the CEFAS Fish Health Inspectorate (FHI) reports of severe eye pathology and blindness in sea and during 1996 and 1997 were investigated. Histopathological findings indicated that the early stages involved degenerative changes to the cornea resulting in various degrees of opacity. However, most samples examined were of more advanced cases where the eyeball had ruptured. In these cases marked necrosis and and inflammation associated with secondary bacterial invaders made it impossible to determine the original cause of the lesion. There were very few reported cases in 1998/99.

CSG 15 (Rev. 12/99) 1 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

Examinations for parasites found in imported cold-water ornamental fish were conducted. The aim was to identify parasites occurring in these fish in order to asses their pathogenicity and to determine whether suitable alternate hosts, necessary for completion of life cycles were already present within the UK. This information being essential to assess the potential threat to indiginous fish stocks. By far the most parasite were detected in fish that were believed to have been collected from wild sources. In contrast, farm bred fish such as koi carp showed very few parasites species to be present. No parasite species known to be specific pathogens to native fish were detected and it was concluded that the actual risk of introduction and establishment of new species of fish parasites to the UK was relatively small. However, surveillance should continue in order to maintain awareness of potential parasite pathogens being introduced via imports of cold- water ornamental fish.

Disease epizootics were investigated in liaison with the FHI and the EA. Bullheads from the River Allen exhibiting external cysts were examined histologically and were found to contain spores of a Dermocystidium species. Host reaction was usually mild , but in some cases an inflammatory response was present. The exact identity of the parasite and its relationship to the salmonid infecting species requires clarification. An epizootic affecting pond-reared carp was found to be caused by the myxosporean parasite Sphaerospora molnari. Severe gill pathology was noted associated with mortalities. This was believed to be the first record of this parasite in the UK.

A survey of the parasite fauna of juvenile salmonids was undertaken with the FHI. Special attention was given to the prevalence of proliferative kidney disease (PKD) and other renal myxosporeans since this information from wild fish was almost totally lacking. Macroscopic signs of PKD were noted in brown trout with salmon also being affected by the disease in several rivers. Histological examination of tissues revealed the presence of the causative agent (Tetracapsula bryosalmonae) in most trout but fewer of the affected salmon. Many fish were found to be infected with another myxosporean parasite Chloromyxum sp. (possibly an undescribed species) but the host response to this species was generally minimal. A Sphaerospora sp. was also recorded. Whether these parasites affect the development or severity of PKD requires clarification. However, the sporogonic stages do not elicit a strong host reaction.

For the full parasite survey a total of 215 trout, 160 salmon, 16 charr and 5 grayling were examined. Twenty- six parasite taxa were found in the four species of juvenile salmonids examined, comprising seven myxozoans, two protistans, four monogeneans, two digeneans, three cestodes, five nematodes and three acanthocephalans. Protista were only recorded in trout and salmon, trout having a more diverse myxozoan fauna. The ciliate Spironucleus barkhanus was found in the gall bladders of two brown trout in Cornwall. This being the first record of this parasite in the UK. This is a significant finding since it is known that this parasite can be highly pathogenic in salmon and other salmonids. However, pathogenicity in native brown trout is unknown. Discocotyle sagittata and Gyrodactylus derjavini were found in two samples of salmon. Crepidostomum farionis was present in all four species of fish and in most rivers. Cestodes were only found in the small sample of charr. Nematodes were present in trout, salmon and charr, with Cystidicoloides ephemeridarum found at high prevalences. Trout, salmon and charr were hosts for the acanthocephalan Echinorhynchus truttae; grayling were hosts for Acanthocephalus anguillae (a new host record), and Pomphorhynchus laevis. No copepods were found despite extensive searches.

Examples the various lesions and parasite specimens encountered during this project were lodged in the Registry of Aquatic Pathology (RAP) collection housed at CEFAS Weymouth. The collection contains examples of pathological material from a wide range of fish and shellfish, mainly based on histological material and is used both for reference and training purposes. The scope of the collection was increased to incorporate parasite specimens, updated to a database format and all records were transferred to the new database. Representative images have been incorporated to be available alongside the other specimen information and it is planned that eventually, the RAP will be available as a CD ROM.

CSG 15 (1/00) 2 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

Scientific report (maximum 20 sides A4)

The main aim of this project was to provide authoritative scientific advice to MAFF on the significance and importance of parasites and diseases in native wild and farmed fish stocks. In particular to provide assessments of the significance of emergent disease problems in order to underpin policy decisions on preventing serious fish disease and in wild freshwater fish. Initially the project was conducted on an opportunistic basis responding in particular to investigation of specific disease outbreaks, often in conjunction with the Fish Health Inspectorate (FHI). However, student placements were used to provide additional resource and were instrumental in allowing more in depth studies to be undertaken on specific topics. Their contribution is indicated in the text and the full reference to their work is included in the list of outputs provided at the end of this report. The main scientific objectives of this project were as follows: -

1. Investigate and assess the threat of introduction of exotic pathogens via the importation of cold- water ornamental fish.

2. Investigate the occurrence of epizootics in wild freshwater fish populations and provide authoritative assessments of their significance.

3. Provide scientifically based information and advice to underpin policy decisions on preventing serious fish disease and parasitism in wild freshwater fish.

During the lifetime of this project it was possible to address several issues concerning disease in wild fish populations and the work programme centred around four main themes. These themes were essentially independent of each other but provided scientific information on a wide variety of conditions either occurring in wild fish populations or of potential threats to native stocks. These themes will be dealt with separately in this report.

A) Examination of cyprinid fry from a variety of river systems for significant parasitism and, in liaison with the Environment Agency (EA) investigate possible effects on recruitment.

This work was initiated because of concerns about the possible decline in wild fish populations in a variety of river systems. Although the factors were unknown it was considered possible that disease may play an important role as a factor affecting successful recruitment of juvenile fish to the adult population. Several years ago small samples of deformed chub fry from were submitted to the CEFAS Weymouth Laboratory for examination. By assessing these histologically it was discovered that the deformities were caused by a myxosporean parasite affecting the vertebral column. Initial studies showed that the parasite was a Myxobolus species (possibly M. ellipsoides) and that the aggressive host response to the parasite was responsible for spinal curvature or scoliosis. Consequently it was decided to examine additional samples comprising several species of fish fry from several river systems in northern England and in the Thames regions (Figure 1 & 2). This work was undertaken with the EA and utilised material collected during their annual fry surveys. The main species examined were chub (Leuciscus cephalus), dace (L. leuciscus), minnow (Phoxinus phoxinus) and roach (Rutilus rutilus). Additional fish species were also examined histologically but were found in general not to be significantly affected by the parasite. However, recent re- evaluation of some of this material has shown that these fish were also affected by additional myxosporeans and other parasite groups and that some of these infections elicited significant host responses.

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Figure 1 Rivers sampled in the Yorkshire / Northumberland Region

Figure 2 River Thames and River Cherwell

4 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

The general methodology used was to obtain whole fry preserved in formalin and to prepare these histologically using conventional techniques. Wax embedded 3 to 5 micron thick longitudinal sections of fry were prepared using rotary microtomes and were stained using Haematoxylin and Eosin (H&E) for general histo-morphology and evaluation of pathological responses. A few additional sections from representative specimens showing typical pathology were also stained with a variety of histological techniques including Gomori trichrome for connective tissue. Sections were also stained with Giemsa stain in order to demonstrate more clearly the parasites and in particular the spores. Using this technique the structure of the spore wall and polar capsules contained within could clearly be discriminated, indicating the parasites in the muscle and vertebral column cysts to be a species of Myxobolus. This was however, insufficient to positively identify the parasite species involved. For this samples of the cysts were examined. Dissection was carried out under stereo microscope to identify and isolate cysts, which were then squashed to release the spores. These preparations were then observed using Nomarski interference contrast or phase contrast microscopy and measurements were taken of the spores in various orientations for taxonomic purposes. The data obtained indicated that the parasite most closely resembled Myxobolus cyprini (Table 1). This species is a well-known parasite of cyprinids which has occasionally been reported as a cause of disease in adult fish in mainland . However, it is extremely difficult to positively identify this species based on spore morphology since it is known that the spore is very variable in shape depending on tissue location and host species. Nevertheless, for the purposes of this report we refer to the muscle cyst parasite as M. cyprini.

Table 1. Comparison of measurements of Myxobolus cyprini (Doflein 1898) from published data and from fish sampled the Yorkshire and Thames regions (all measurements in µm).

Spore Published data for Cysts A Cysts B Cysts C measurements M. cyprini

Length 10.0 - 13.5 11.43 10.83 12.07 Width 7.6 - 9.7 8.30 7.69 8.50 Length LPC* 5.6 - 6.9 4.42 4.48 5.47 Width LPC 3.1 - 4.3 2.56 2.42 2.82 Length SPC** 5.0 - 6.0 3.18 3.26 3.83 Width SPC 2.7 - 3.7 2.20 2.02 2.36

Measurements of spores from cysts A & B were from fish obtained from the Thames Region and those from cysts C are from fish obtained from the Yorkshire region. * Larger polar capsule, **smaller polar capsule.

Examination of stained tissue sections of whole fry revealed that in many samples that appeared macroscopically normal, numerous myxosporean cysts were present in the musculature. In addition, in some of these fish the cysts were particularly numerous and elicited a severe inflammatory reaction, especially where the cysts had ruptured. This intense inflammatory reaction was considered severe enough to result in the mortality of affected fish, particularly where cysts were located close to the skin surface where ulceration could occur. In order to evaluate the sectioned material a simple method for the assessment of intensity of infection was devised based on the number of cysts observed in each section of fry (see Table 2). This assessment was generally based on examination of a single longitudinal histological section.

The highest prevalence of infection occurred in species obtained from the Thames catchment area. However, samples from this region were only collected for two years and it was not possible to assess if fish were consistently parasitised at high prevalence rates. This would seem to be unlikely since samples from the Yorkshire region demonstrated variable prevalence over a period of years.

5 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

Table 2. Assessment of severity of myxosporean infections.

Grade No. of cysts per fish (from histological section) 0 0

1 1 - 4

2 5 - 9

3 10 - 20

4 > 20

During the lifetime of this project samples of fry were assessed annually in order to obtain temporal trend data for the four target fish species. This data was most comprehensive for the Yorkshire region and demonstrated fluctuating levels in both prevalence and intensity in samples collected since 1993. The reasons for these fluctuating levels remain uncertain but water quality and particularly pathogenicity of the parasites are likely to play an important role. This work is continuing in liaison with the EA and is providing unique data on the distribution and prevalence of parasites in cyprinid fry in a number of river systems. This information forms one aspect of the larger initiative concerning factors affecting cyprinid recruitment. Although the data still need to fully analysed, it is already clear that there is a need to more fully understand the life cycle requirements of the parasites involved. This aspect has been addressed in another MAFF- funded project on ‘Life cycles of myxosporean parasites including the causative agent of proliferative kidney disease (FC1112). One aspect of that work has concentrated on the role of oligochaetes as alternative hosts necessary for the transmission of these parasites to fish. Since it has been established that for several species of Myxobolus infecting freshwater fish the infective stage is an actinospore, released from oligochaetes, it is probable that a number of biotic and abiotic factors affecting oligochaete populations will in turn influence the prevalence and intensity of parasitism within the fish host. Many of these factors and their impact remain unknown.

B) Investigate reports of severe eye pathology in sea trout and salmon

There were a few cases of eye pathology problems in wild salmon and sea trout reported in 1996 and 1997 in fish from south coast rivers. Although histopathological changes were present in these fish, it was not possible to establish the aetiology. The main changes observed included degenerative changes to the cornea and which appeared to progress in more advanced cases, eventually resulting in rupture of the orbit. Most cases submitted for examination were of such cases and the marked inflammation and necrosis present, often associated with secondary bacterial invaders made it impossible to determine the original cause. Subsequent monitoring in conjunction with the Fish Health Inspectorate (FHI) and EA staff, of stocks of wild salmon captured for stripping have shown some eye pathology but it could not be confirmed that this was the same as that seen in wild caught fish. The significance of these cases also remains obscure.

C) In liaison with the Fish health Inspectorate, undertake a survey of cold-water ornamental fish imports for the presence of parasite pathogens. Evaluate potential threat to native wild and farmed stocks.

Examinations for parasites found in imported cold-water ornamental fish were conducted. The aim was to identify parasites occurring in these fish in order to assess their pathogenicity and to determine whether suitable alternate hosts, necessary for completion of life cycles were already present within the UK. This

6 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

information being essential to assess the potential threat to indigenous fish stocks. Much of this work formed the basis of a work placement project (P A Wright).

Cold water ornamental fish from various origins were subjected to full parasitological examinations using a standardised protocol. Gill and skin scrapes were taken and samples of each organ and tissue was taken and examined using phase contrast optics. The gills and digestive tract were also examined under a dissecting microscope. Parasites were photographed and measured for taxonomic purposes. Tissues were also examined histologically using conventional processing techniques and a variety of staining techniques.

A very low prevalence of parasites were found in imported carp species and it presumed that most fish had been treated before transportation and that this had removed most of the parasite fauna originally present. From other fish species a wide range of parasites were found in fish entering the UK. Species from the parasite classes Cestoda, , Nematoda, Acanthocephala, , Crustacea, Ciliata and were all encountered (see Table 3).

Myxosporeans were found in the spleen and the kidney of bitterling imported from Hong Kong. These were identified as Myxidium rhodei and Myxobolus cyprini. Both of these species are known to be present in the UK. Myxidium rhodei is commonly found in roach, Rutilius rutilius. The primary organ of infection is the kidney where in heavy infections severe damage can occur. The number of glomeruli may decrease, atrophy of surrounding tissues may occur and granulomatous inflammatory changes can result in the renal interstitial tissues. Myxobolus cyprini is frequently found in cyprinid in Europe, China and . A species of Acanthocephala, possibly of the genus Pallisentis was found in the intestine of weatherloach imported from Hong Kong. This parasite has not been recorded in native fish species.

The monogenean vastator was found on the gills of goldfish imported from China. D.vastator generally only infests fish that are smaller than 3-5 grams. The occurrence of the related species D. extensus and D. vastator are thought to be possibly widespread on carp in the British Isles. Since the optimal conditions for development of D. extensus are temperatures of between 13°C and 15°C and British waters regularly reach these temperatures at times during the summer period, spread of the D. vastator is likely.

Protistan parasites such as ciliates were commonly encountered in several of the fish species examined. These were often associated with the external surfaces of the fish and were not associated with macroscopic pathological changes. Although it was not possible to identify to species the trichodinids found, it was considered that these parasites were likely to be present on fish already in the UK. The trichodinid detected in the renal tubules of koi carp that had originated from Israel also did not appear to be associated with a significant host response.

By far the most parasite species were detected in fish that were believed to have been collected from wild sources. In contrast, farm bred fish such as koi carp showed very few parasites to be present. This is probably explained by the fact that fish are reared in semi-controlled environments and are usually treated before transport. Although only relatively few samples became available for examination during the course of this project, no parasite species known to be specific pathogens to native fish were detected. It was concluded that the actual risk of introduction and establishment of new species of fish parasites to the UK was relatively small. However, surveillance should continue in order to maintain awareness of potential parasite pathogens being introduced via imports of cold-water ornamental fish.

7 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

Table 3. Parasite isolated from imported ornamental fish.

Fish Species Origin Parasites found Location Bitterling (Rhodeus Hong Kong Myxobolus cyprini Spleen and Kidney ocellatus) Myxidium rhodei Weatherloach Hong Kong L1 & L2 nematode Intestine (Misgurnus fossilis) (batch A) Unidentified Kidney myxosporeans Thelohanelus. sp. Gills Myxobolus.sp Bile, brain, kidney Digenean Kidney metacercaria Chloromyxum. sp. Bile Weatherloach Hong Kong Digenean Kidney, muscle (Misgurnus fossilis) (batch B) metacercaria Nematodes Intestine Thelohanelus. sp. Kidney, gills, liver, intestine Myxidium. sp. Kidney Pallisentis? sp. Intestine Myxobolus. sp. Epidermis, liver, kidney Goldfish (Carassius Local pond White spot Skin, gills auratus) Trichodinids Gills Dactylogyrids Gills Goldfish (Carassius China Gills auratus)

Koi carp (Cyprinus Israel Gyrodactylus. sp. Gills carpio) Dactylogyrus. sp. Gills Trichodinids Renal collecting ducts Khuli loach Myxosporeans Renal tubules (Acanthophthalmus Nematode. sp Adipose tissue kuhli)

D) Assess the importance of other diseases and parasites of wild freshwater fish. i) Epizootic of Dermocystidium sp. in bullheads (Cottus gobio L.) in southern England.

Bullheads with epidermal cysts on the skin and fins and measuring up to 2 to 3 mm in diameter, were recorded by the Environment Agency from the River Allen and tributaries in southern England since 1992. Infection prevalence rates of over 50% were recorded. Samples of similar infections were obtained during August 1996 by a member of the Fish Health Inspectorate. Examination of the cyst contents revealed the presence of numerous protistan ‘spores’, typical of the genus Dermocystidium and measuring approximately 7µm in diameter. Several examples of ruptured cysts were seen and in some of these cases the epithelium was ruptured allowing release of spores to the environment. The infection was considered unlikely to be lethal. Similarities between the Dermocystidium in bullheads and species known to infect salmonids have yet to be ascertained. ii) Detection of Sphaerospora molnari affecting carp

8 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

In liaison with the EA the parasite Sphaerospora molnari was identified as the cause of an epizootic in pond reared carp. The parasite caused significant damage to the gills of affected fish with most of the respiratory epithelium being replaced with developmental stages and spores of the parasite. This epizootic appears to have been the first record of the parasite in the UK. Samples of material from this epizootic have been placed in the Registry of Aquatic Pathology (RAP) collection held at CEFAS Weymouth. iii) Investigations into the parasite fauna of wild salmonids in England and Wales.

As part of the routine monitoring of the health status of wild fish populations a survey of the parasite fauna of juvenile salmonids in British rivers was undertaken with the FHI. The aim was to establish the degree of parasitism present in these salmonids and to assess whether specific parasites were inducing a pathological response. Because information on the prevalence of PKD in wild freshwater salmonids was almost totally lacking, particular attention was given to the presence of renal myxosporean infections with PKX (causative agent of Proliferative Kidney Disease (PKD)) and Chloromyxum sp. The parasite survey formed the basis of a work placement project (E Smith).

Fish were collected from 26 sites on 23 rivers in England and Wales, mainly by electrofishing, between June and September 1997. Pectoral and dorsal fins were removed on site, placed in a small amount of water and maintained at +4°C until examination in the laboratory. Samples for microbiological investigation were routinely taken from visceral organs and the fish were then preserved in neutral buffered formalin (NBF) for a minimum of 24 hours. In the laboratory, fish were soaked overnight in distilled water prior to examination. NBF and water containing the fish was passed through a fine filter and examined for the presence of parasites. Total length, fork length, total weight and gutted weight were measured for each fish. Skin, fins, gills, nares and buccal cavity were examined under a stereo microscope and smears taken of all these organs. Samples of visceral organs were examined under stereo microscope and at x100 and x400 using conventional phase contrast microscopy. Kidneys from all fish were removed and prepared for wax histology using standard protocols. 5µm sections were stained using Giemsa and H&E and examined for the presence of myxozoan parasites. Parasites were identified from published host-parasite lists, published descriptions and by comparison with samples in the Registry of Aquatic Pathology (RAP) held at CEFAS Weymouth. Images of parasites were stored on a Lucia imaging system (Nikon UK Ltd) or photographed conventionally. Representative examples of all parasites were kept in NBF. The measures of infection were (i) prevalence, defined as the number of fish infected with a particular parasite divided by the total number of fish in the sample, expressed as a percentage, and (ii) abundance, defined as the total number of individuals of a particular parasite species in a sample of hosts divided by the total number of individuals of the host species in the sample.

Renal myxosporidiosis in salmonids.

Early findings of trout and salmon with clinical signs of proliferative kidney disease (PKD) but no evidence of infection with the PKX organism (now named Tetracapsula bryosalmonae) were confirmed in several river systems. Infections with an unidentified Chloromyxum species were found in several of these cases and infections with this parasite were found to be widespread. Histological studies were undertaken in order to assess the pathological effect of the organism. The results confirmed previous findings that the parasite appears to cause only minimal damage to the renal tubules and little evidence could be found of extrasporogonic stages which could have been responsible for the inflammation giving rise to the clinical signs in the fish. The Chloromyxum was morphologically similar to a well-recognised pathogenic species (C. majori) infecting North American salmonids (see Table 4). However, the characteristic glomerular pathology associated with C. majori infections was not observed in any of the specimens examined in the present study and spore morphology based on light microscope findings could not confirm the identify of the parasite as C. majori. Scanning electron microscopy was attempted in order to characterise the parasite in UK salmonids. This technique is required in order to visualise the surface morphology of the mature spores. The number and pattern of surface ridges being critical taxonomic features. This work is still in progress.

9 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

Table 4. Comparison of measurements of Chloromyxum sp. spores from the renal tubules of salmon and brown trout with published data of Chloromyxum majori from steelhead trout from N. America. All measurements are given in microns.

Parasite from salmon (S. Parasite from Measurements of salar) Brown trout (S. trutta) Chloromyxum majori from N. American steelhead trout (O. mykiss). Reference Present study Present study Yasutake & Wood, 1957. Spore length 7.4 (6.9 - 7.8) 7.7 (7.4 - 7.9) 5.6 – 7.4 Spore thickness 7.4 (6.4 - 8.0) 7.5 (7.2 – 7.8) 5.6 – 7.4 Spore width 7.6 (6.9 - 8.0) 7.8 (7.6 – 8.0) 5.6 – 7.4 Length of small 2.5 (1.9 – 3.4) 2.5 (1.9 – 3.3) 2.0 – 3.6 polar capsule Width of small 2.4 (2.0 - 2.9) 2.4 (2.2 – 2.8) 2.0 – 3.6 polar capsule Length of large 2.8 (2.1 - 4.4) 2.8 (2.1 – 3.1) 2.0 - 3.6 polar capsule Width of large 2.7 (2.3 - 3.5) 2.5 (2.3 - 3.1) 2.0 - 3.6 polar capsule

The Chloromyxum sp. parasite was widely distributed in 20 of the 23 river systems sampled in England and Wales. However, in addition to the Chloromyxum sp. found in both trout and salmon, several fish were found to be infected with a Sphaerospora sp. or exhibiting clinical signs of PKD (Table 5). A few fish were found to harbour multiple infections. Further cases without clinical symptoms of PKD were found to be infected with T. bryosalmonae by examination of stained histological sections of renal tissue. The significance of these multiple infections is still unclear, but pathology associated with the both the Sphaerospora sp. and Chloromyxum sp. was minimal and was restricted to localised changes to the epithelium of the renal tubules. No evidence was found of extrasporogonic stages of these parasites, which are likely to elicit a more vigorous host response.

Table 5. Prevalence of renal myxosporidiosis in salmonids.

Salmo salar trutta N = 70 N = 210 (%) (%) Macroscopic PKD 10.0 18.1 Renal T. bryosalmonae cells 4.3 14.3 Sporogonic Sphaerospora sp. 1.4 0.9 Sporogonic Chloromyxum sp. 48.6 30.0 Overall prevalence of 83.3 95.6 Chloromyxum sp. in the rivers sampled.

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Full parasitological survey of salmonids.

A total of 160 salmon, 215 brown trout, 16 charr and 5 grayling were sampled between June and September, 1997 and examined for parasites. A total of 26 different species of parasites were recorded and identified. All parasite phyla with the exception of Microspora and Arthropoda were represented (Table 6). The results showed an uneven distribution of parasites in the rivers sampled. For example, five protistan parasites were recorded from the River Avon but none from the River Frome.

Table 6. List of parasite species recorded from wild salmonids from England and Wales. Site of infection in the host, overall prevalence and range ( ) are indicated.

Parasite Species Location in Fish Host Host Trout Salmon Charr Grayling PROTISTA Chloromyxm cf majori Kidney tubles 27 22 - - Chloromyxum truttae Gall bladder 1 - - - Dermocystidium sp. Gills 1.9 1.9 - - Henneguya zschokkei Gills 1 - - - Myxidium truttae Gall bladder, Liver 4 1.9 - - Myxobolous neurobius Brain, spinal 3.7 - - - column Tetracapsula bryosalmonae (PKX) Kidney 11 1.25 - - Sphaerospora sp. Kidney - 0.6 - - Spironucleus cf barkhanus Gall bladder 1 - - - MONOGENEA Discocotyle sagittata Gills - 1.9 (0-1) - - Gyrodactylus derjavini Skin, fins 7 of 18* 2 of 13* - - G. thymalli Skin, fins - - - 1 of 1* G. truttae Skin, fins 1 of 18* - - - DIGENEA Crepidostomum farionis Intestine, stomach, 35 (0-16) 32 (0-50) 13 (0-10) 20 (0-3) pyloric caeca, Gall bladder Diplostomum spathaeceum Eyes 19 (0-25) 4 (0-6) - - CESTODA dentriticum Viseral cavity - - 46 (0-9) - Eubothrium crassum Intestine - - 56 (0-2) - Proteocephalus cf. neglectus Intestine - - 44 (0-20) - NEMATODA Camallanus lacustris Stomach 1 (0-9) - - - Cystidicola farionis Swim bladder 4 (0-35) 1.9 (0-1) 6 (0-1) - Cystidicoloides ephemeridarum Stomach, intestine, 37 (0-65) 36 (0-60) - - pyloric caeca Pseudocapillaria salvelini Intestine - 1.3 (0-3) - - Raphidascaris acus Intestine - 0.6 (0-1) - - ACANTHOCEPHALA Acanthocephalus anguillae Intestine - - - 20 (0-1) Echinorhynchus truttae Intestine 20 (0-21) 7.5 (0-8) 6 (0-1) - Pomphorhynchus laevis Intestine - - - 40 (0-1) TOTAL NUMBER OF FISH EXAMINED 215 160 16 5 *Number of rivers infected out of a total number of rivers sampled for Gyrodactylus spp

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Salmon (Salmo salar) parasites.

From salmon, four species of myxosporeans were found, most commonly Chloromyxum sp. within renal tubules (see above). Myxidium truttae was recorded in the gallbladder and in heavy infections in the liver at low prevalences in two rivers, the River Lyd and River Allen. Both of these rivers being located in Cornwall. Dermocystidium sp. was found encysted in the gills of salmon (and brown trout) from three river systems. These cases were not associated with macroscopic changes to the gills and cyst numbers were low.

The monogenean gill parasite Discocotyle sagittata was found in salmon from the River Tavy and Eastern Cleddau at prevalences of 10% and 33% respectively. The only other monogenean parasite detected during the survey was Gyrodactylus derjavini, which was recorded from the pectoral fins of fish from the Rivers Allen and Tavy. Two digeneans, Crepidostomum farionis and Diplostomum spathaceum were found. C. farionis was one of the commonest parasites found infecting salmon, in 7 of the 13 river systems and at prevalences of up to 83%. The main sites of infection were the stomach, pyloric caecae and intestine. Occasionally it was found in the gallbladder. D. spathaceum was found in the eye of salmon from three river systems at prevalences between 14% and 80%.

Four different nematode species were recorded from salmon. Cystidicoloides ephemeridarum was the commonest species found with a prevalence of up to 92%. It was found in the digestive tract, sometimes in large numbers (60 worms were found in a single fish from the River Lyd). Cystidicola farionis located in the swimbladder were found in fish from the Rivers Allen and Frome in southern England. The nematodes Pseudocapillaria salvelini and Raphidascaris acus were found only in salmon from the River Lyd. The site of infection for both species was the intestine.

Echinorhynchus truttae was the only acanthocephalan recorded in salmon. Prevalences ranged from 12% to 44%.

Brown trout (Salmo trutta) parasites.

The only ciliate parasite recorded from brown trout was Spironucleus cf. barkhanus, a previously unrecorded parasite both in the UK and from brown trout. It was found infecting the gallbladder. Several myxosporean parasites were found, including M. truttae at prevalences of up to 25%. Henneguya zschokkei and Myxobolus neurobius were found rarely.

The only monogenean parasite detected was G. derjavini, which was more widespread in brown trout than salmon, occurring in 7 out of the 18 river systems sampled for brown trout. As in salmon, Crepidostomum farionis was the commonest parasite found affecting up to 80% of the fish sampled at certain locations. The sites of infection were also the same as in salmon. D. spathaceum was also found in brown trout at prevalences ranging from 11% to 80%. At certain locations the nematode C. ephemeridarum was found in all fish examined and although large numbers of worms were occasionally found in individual fish, little pathology was evident. Cystidicola farionis was only detected in fish from 4 river systems. An additional nematode species, Camallanus lacustris was found in fish from two river systems at prevalences of 10 and 11%.

The only acanthocephalan recorded in brown trout was E. truttae, from six river systems at prevalences between 13% and 100%. No pathology was noted with these infections.

Charr (Salvelinus alpinus) parasites.

Charr were obtained from one site, Lake Windermere. Fewer parasite species were recorded in charr than in either salmon or brown trout. Crepidostomum farionis was the only digenean parasite found, present in two out of sixteen fish examined. A single nematode (Cystidicola farionis) was found in one fish. Charr were the only fish species examined which harboured cestode parasites. Diphyllobothrium dentricum was found in the visceral cavity of seven fish with up to nine parasites per fish. Eubothrium crassum was detected in the intestine of nine fish with one or two parasites present in each. A third species (Proteocephalus neglectus)

12 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

was also recorded in the intestine of seven fish. A single specimen of the acanthocephalan E. truttae was also recorded.

Grayling ( thymallus) parasites.

Only four species of parasite were recorded from the small number of grayling that were available for examination. These are indicated in Table 6. A significant finding of the acanthocephalan Acanthocephalus anguillae appears to be a new record for grayling.

Conclusions

In summary, twenty-six parasite species were recorded during the survey. The Protists were represented by nine species and the digeneans by only two species. Brown trout contained the greatest number of parasite species, sixteen in total; salmon, fourteen species; charr, six species and grayling only four parasite species. There was a discontinuous distribution of parasites in the wild salmonid populations in England and Wales. The ciliate Spironucleus cf. barkhanus had not previously been recorded previously in the UK or in brown trout. Further samples will be required in order to verify this finding and confirm the specific identity of the parasite. In particular it is important to determine whether this parasite is capable of causing significant pathology in infected fish since S. barkhanus is recognised as a significant pathogen of salmon.

The monogenean G. thymalli was only found in grayling supporting the view that this species does not affect either brown trout or salmon. Similarities between this species and the serious salmonid pathogen G. salaris have caused concern for diagnosticians and significant resources have been allocated to the development of robust diagnostic techniques for the identification of this species. D. sagittata was commonly found although it was not associated with significant pathology in wild fish. In contrast, its presence in farm situations can cause problems, primarily because it has a direct life cycle and the free-swimming oncomiracidia achieve sexual maturity within only two months.

Most of the parasites found did not appear to elicit a serious pathological response in the host. Exceptions were the myxosporean T. bryosalmonae and to a lesser degree, the Chloromyxum sp. infecting renal tubules. However, the kidney was the only organ routinely examined histologically and this may be one reason why , the causative agent of whirling disease was not detected. Although it is recognised that most parasites are capable of inducing a vigorous host response, little indication based on macroscopic examination of this was found during this survey. Additional investigations including examination for histopathological lesions are required to provide data on the significance of the parasites found to the hosts.

It is clear that a large number of factors affect the parasite fauna of fishes and this has been utilised in a number of studies using parasites as indicators of environmental change, including anthropogenic pollution. Poor water quality can favour certain parasites such as monogeneans, but for others it may be detrimental and for example result in the reduction in numbers of intermediate hosts required to complete parasite life cycles. The influence of biotic and abiotic factors should be taken into account in the assessment of parasite data obtained in future surveys.

E) Establishment of the Parasitology section of the Registry of Aquatic Pathology (RAP).

Although not specifically identified as one of the main objectives of this project. Many of the specimens examined during the course of these studies have either been deposited in the RAP or are being prepared for accession. Because of the increasing attention being given by MAFF to significant fish and shellfish parasite pathogens it was decided to develop further the scope of the RAP to include parasite specimens as well as slides of histological specimens. For this, the efforts of a work placement student (Man Tsuey Tse) are gratefully acknowledged. The RAP comprises a unique collection of pathological and parasitological specimens from fish and shellfish. There are currently over 800 accessions archived and this number is continually increasing. The RAP was developed to facilitate access to the numerous specimens accumulated over the years at CEFAS Weymouth and was organised according to host groups, for example, cyprinids and salmonids. However, during 1999 a database format for the RAP was designed and all records were

13 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

transferred to the new database. The intention being to facilitate the use and perusal of the collection. In addition, representative images were incorporated to be available for viewing together with the other details associated with each specimen. This work is still at an early stage and is ongoing. It is intended that eventually, the RAP will be available on a CD ROM and possibly be made available to interested parties via the Internet. Although the RAP database has been constructed, the process of verification and updating based on new scientific information is ongoing. The value of the RAP as a source of reference for MAFF and the wider scientific community is increasing as the number of accessions become incorporated continues. It is anticipated that this will be a continuing trend.

Outputs

These included published scientific papers, conference proceedings, presented papers and posters and placement student reports.

Feist, S. W. (1997) Pathogenicity of renal myxosporidiosis in fish. Bulletin of the EAFP, 17 (6), 209-241.

Feist, S. W. (1999) Effects of PKD on wild fish populations in the U.K. MAFF/CSG Workshop on Proliferative Kidney Disease (PKD) in wild and cultured salmonids. CEFAS Weymouth Laboratory, 15th – 17th November, 1999.

Feist, S. W., Dampier, K. & Frear, P. Myxosporidiosis in juvenile cyprinids. Poster presentation, EAFP 8th International conference, September 1997.

Feist S. W., Hurrell R. H. and Longshaw M.. Observations on an epizootic of Dermocystidium sp. in bullheads (Cottus gobio L.) from a river in southern England. Oral presentation. British Society of Parasitology, April 1997.

Feist, S. W., Hurrell, R. H. & Longshaw, M. Observations on an epizootic of Dermocystidium sp in bullheads (Cottus gobio L.) from a river in southern England. Poster presentation, EAFP 8th International conference, September 1997.

Feist, S W & Longshaw, M. Structure and development of Myxobilatus gasterostei (Parisi, 1912) (), a parasite of the kidneys of the stickleback Gasterosteus aculeatus. Poster presentation, British Society for Parasitology, April, 1998.

Feist, S. W., Longshaw, M. & Frear, P. (1999) Myxozoan parasites of cyprinid fry - is there an effect on development and recruitment? Int. Symp. of the Fisheries Soc. of the British Isles. 5-8th July, 1999, University of St Andrews, Scotland.

Feist, S. W., Smith, E. S. & Longshaw, M. (1999) Inter-relationships between PKD and infections with Chloromyxum sp. in juvenile salmonids. British Soc. Of Parasitology, Spring Meeting, 11th –14th April, 1999. University of Warwick.

Hill, B. J. (1999) Databases in fish health management. Bulletin of the EAFP, 19 (6), 279-281.

Lom, J., McGeorge, J., Feist, S.W., Morris, D & Adams, A. (1997) Guidelines for the uniform characterisation of the actinosporean stages of parasites of the phylum Myxozoa. Diseases of Aquatic Organisms, 30, 1-9.

14 Project Characterisation and pathogenesis of epizootics in wild MAFF FC1106 title freshwater fish (Phase II) project code

Longshaw, M. & Feist, S. W. Pathogenic parasites of marine aquarium fishes. Poster presentation, British Society of Parasitology, April 1997

Longshaw, M & Feist, S W. Oligochaetes as hosts for aquatic parasites. Poster presentation, EAFP 8th International conference, September 1997.

Longshaw, M & Feist, S. W. Studies on the actinosporean fauna of oligochaetes in river systems in southern England. Oral presentation, British Society for Parasitology, April, 1998.

Morris D. J.., Adams A.., McGeorge J. and. Feist S. W. Immunohistochemical analysis of wild and cultured fish for PKX the causative agent of proliferative kidney disease. Oral presentation. British Society of Parasitology, April 1997.

Smith, E. S. Investigations into the parasite fauna of wild salmonids in England and Wales. SCS project report. Sept. 1997 to Sept. 1998.

Smith, E, Gardiner, R, Feist, S. W. & Longshaw, M. (1999) A survey of parasites of juvenile salmonids in England and Wales. Poster presentation British Soc. for Parasitol. Exeter, April 1999.

Tse, M. T., Development of the parasitology section of the Registry of Aquatic Pathology (RAP) including studies on selected parasites. SCS project report. Sept. 1998 to Sept. 1999.

Tse, M. T., Feist, S. W. & Longshaw, M. (1999) Studies on Sphaeromyxa spp. (Thelohan, 1892) (Myxozoa) from selected fish species. 5th Int. Symp. on Fish Parasites. 9-13th August, 1999. Czech Republic.

Wright, P. A. Parasites in imported cold-water ornamental fish. SCS project report. Sept. 1996 to Sept. 1997.

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